Declined plasma microfibrillar-associated protein 4 levels in acute coronary syndrome

Background Microfibrillar-associated protein (MFAP4), initially identified as an extracellular matrix protein, has been demonstrated in multiple human disorders, but it is yet to be discovered following acute coronary syndrome (ACS) in clinical practice. Therefore, this study aimed to investigate the relationship between circulating MFAP4 levels and coronary stenosis in ACS. Methods We performed the study in 148 ACS subjects, including 75 ST-segment elevation myocardial infarction (STEMI), 27 non-ST-segment elevation myocardial infarction (non-STEMI) and 46 unstable angina (UA). Clinical variables were collected and Gensini and Syntax stenosis scoring systems were applied to assess the severity of coronary stenosis. Kaplan–Meier and logistic regression analysis were used to analyze the relationship between MFAP4 and the severity of coronary stenosis or ACS outcomes. Spearman analysis was used to describe the correlation between MFAP4 and clinical parameters. Results Circulating MFAP4 levels were significantly decreased in the STEMI group (0.008 ng/ml) compared with the non-STEMI group (0.014 ng/ml) and UA group (0.019 ng/ml) (p < 0.001). After adjusting for confounding factors, we found that MFAP4 was an independent risk factor for STEMI (odds ratio = 0.395, 95% CI 0.174–0.895, p = 0.026). MFAP4 level was negatively correlated with Gensini score and Syntax score (r = − 0.311 and − 0.211, p < 0.001 and 0.01, respectively). Based on the MFAP4 level of 0.117 ng/ml, ACS patients were divided into two groups: the low-MFAP4 group (< 0.117 ng/ml, n = 60) and the high-MFAP4 group (≥ 0.117 ng/ml, n = 88). After the median follow-up of 165 days, Kaplan–Meier survival analysis revealed that the MACE-free rate was significantly lower in ACS patients with lower MFAP4 levels (p = 0.009). Conclusions MFAP4 has a potential as a biomarker for the degree of coronary stenosis in ACS. Confirmation of observations in larger cohorts and longer follow-up periods is warranted. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-023-01002-z.


Introduction
Acute coronary syndrome (ACS) is the most common cardiovascular emergency, accounting for nearly half of the global cardiovascular morbidity and mortality, placing an enormous economic burden worldwide [1]. Typically, depending on the range of myocardial ischemia, ACS may present as unstable angina (UA), non-ST-segment elevation myocardial infarction (non-STEMI), and ST-segment elevation myocardial infarction (STEMI). Since ACS may proceed into irreversible myocardial damage, early and precise identification of such urgent situation are crucial. So far, detection of coronary artery stenosis heavily relies on invasive methods, such as coronary angiography. Although there are some biomarkers in identifying myocardial necrosis, including cardiac troponins (cTn) and creatine kinase-MB (CKMB)-the gold standard markers, they cannot reflect the severity of coronary stenosis, since patients in severe vascular condition may need individualized treatment. Thus, it is significant to find novel biomarkers that precisely detect myocardial damage and also reflect the severity of coronary stenosis.
Microfibrillar-associated protein 4 (MFAP4), also named microfibrillar-associated glycoprotein 4 (MAGP4), known as 36-kDa microfibril-associated glycoprotein, is a secreted matricellular protein and belongs to the fibrinogen-related domain superfamily, including fibroleukin, ficolin, angiopoietins, and tenascin, which is initially associated with tissue homeostasis, cardiovascular system development and normal endothelial function [2]. MFAP4, mainly located in elastic fibers and highly expressed in blood vessels, probably contributed to vascular remodeling. Besides large arteries and arterioles, MFAP4 mRNA transcript levels were relatively higher in myocardial tissue and lung [3], which is in agreement with its important role in cardiac hypertrophy, asthma and chronic obstructive pulmonary disease [4]. Recently, animal studies have reported that MFAP4 deficiency inhibited myocardial hypertrophy and myocardial fibrosis, and improved cardiac function [5][6][7]. Notably, some reports even suggested the possibility of using MFAP4 as a biomarker in atherosclerosis-related diseases [3]. Additionally, MFAP4 has been identified with a lower heritability and relatively limited basic variation, implying MFAP4 as an excellent biomarker candidate in reflecting the process of disease induction [8,9]. And, the association between plasma MFAP4 and other clinical parameters and its role in the prognosis of ACS is still under discussion. Accordingly, in this study, we aimed to investigate the correlation between severity of coronary stenosis, calculated by coronary angiography, and the circulating MFAP4 levels in a cohort of patients admitted with ACS.

Study population
In this study, 148 ACS patients, including 75 STEMI, 27 non-STEMI and 46 UA, admitted at the Department of Cardiology, Beijing Chaoyang Hospital from May 2021 to July 2021 were recruited in this study (Fig. 1). Clinical classification of ACS was based on expert consensus statements. All subjects with a history of stent implantation, a history of coronary artery bypass graft, cardiomyopathy, myocarditis, congenital heart disease, chronic liver or kidney disease, systemic or local inflammatory or infectious disease, autoimmune disease, pregnancy, and malignancy were excluded from the study. Written, informed consent was obtained from all subjects before their participation in the study. The study protocol was approved by the Beijing Chaoyang Hospital Ethics Committee and adhered to the Declaration of Helsinki. This study was registered in the Chinese Clinical Trial Registry (No. ChiCTR2100045902).

Laboratory assay
Venous blood samples were drawn in tubes with ethylenediaminetetraacetic acid (EDTA) and centrifuged at 3000 rpm for 10 min at 4 °C. Plasma was stored in a -80 ℃ ultra-low-temperature refrigerator until biochemical analysis. Plasma MFAP4 levels were measured using commercially available microplate ELISA kit (Human Microfibrillar Associated Protein 4 ELISA kit, Abbexa LTD, Cambridge, UK) following the sandwich ELISA principle (Catalog No. abx152321, sensitivity < 0.121 ng/ mL, intra-assay coefficient of variation < 10%, and interassay coefficient of variation < 10%). Other clinical and biochemical parameters were measured by standard procedures.

Evaluation of coronary artery disease severity
Coronary angiographic image was collected from the catheter laboratory during hospitalization, and Gensini and Syntax stenosis scoring systems were applied to assess the severity of coronary stenosis by two independent experienced interventional clinicians. As published previously, the Gensini score was based on the grade of stenosis, and the sum of the scores for individual lesions was defined the severity of coronary artery disease. The Syntax score was an anatomical calculator that quantitatively describes vessels with diameters > 1.5 mm and stenosis > 50% in the light of location, function, complexity and obstruction, and was performed with internet-based SYNTAX calculator (www. syntax. score. com).

Statistical analysis
Normal distribution of data was assessed using the Kolmogorov-Smirnov test. Numerical variables with a normal distribution were presented as the mean ± SE, numerical variables with a skewed distribution as the median (P25, P75). The χ 2 test was performed to analyze demographic data. Comparisons in groups with parametric conditions were performed using Student's t test, and with the Mann-Whitney U test in groups without parametric conditions. Subsequently, analysis was undertaken through multivariate, step-wise forward conditional logistic regression analysis to assess a set of independent variable predictors of ACS. Spearman correlation was used for correlation analyses. The sensitivity and specificity of the MFAP4 level were determined by the receiver operating characteristic curve (ROC), and the best cut-off value was set to maximize the sensitivity and minimize1-specificity. Kaplan-Meier survival curves were applied to depict survival curves and a logrank test was used to assess survival differences between groups. Clinical events were followed up since patient's hospitalization, and after discharge medical record and phone interviews help record major adverse coronary events (MACE), including cardiovascular death, rehospitalization, advanced heart failure and revascularization. Values of p < 0.05 were regarded as statistically significant. All statistical analyses were performed with IBM SPSS Statistics 24.0 (IBM Corp, Armonk, NY).

Baseline clinical characteristics of the cohort
The present study included ACS patients with 75 STEMI, 27 non-STEMI and 46 UA. The baseline clinical characteristics are described in Table 1. The mean age of the study population was 61 ± 11 years and 118 (79.7%) participants were male. Regarding demographic data, except for hypertension, which was more common in UA patients, there was no other statistically significant difference regarding age, gender, prevalence of diabetes, hyperlipidemia and arrhythmia among three groups. In the laboratory measurement, with gradually aggravated cardiac function and increased myocardial injury, ACS patients probably faced unregulated abnormal lipid and glucose metabolism, increased inflammatory biomarkers: higher leukocytes (p < 0.001) and higher C-reactive protein (p < 0.001), and accompanied with more renal and hepatic dysfunction (increased creatinine, alanine aminotransferase and aspartate aminotransferase). As shown in Fig. 2 and Table 1, the median MFAP4 levels were 0.133 ng/ml, with an interquartile range of 0.065-0.262 ng/ml. Remarkably, circulating MFAP4 levels were gradually decreasing following the developed degree of myocardial ischemia, which were lowest in the STEMI group (STEMI vs. non-STEMI p = 0.018 and STEMI vs. UA p < 0.001, respectively). Medication on admission and medication on discharge is shown in Table S1.

Association between plasma MFAP4 levels and biochemical variables or coronary stenosis
The relationship between baseline MFAP4 and other clinical biomarkers were analyzed with spearman correlation models. As shown in Table 2 The association between ACS and circulating MFAP4 levels was analyzed in logistic regression models. Although MFAP4 did not confer a role in myocardial infarction (MI) (STEMI + non-STEMI) (p = 0.304), in STEMI patients, declined plasma MFAP4 levels were more common (OR = 0.216, 95% CI 0.070-0.665, p = 0.008, Table 3). When all significant univariate determinants (p < 0.05), including conventional coronary artery disease risk factors like TC, LDL, cTnI, CKMB, were entered into multivariate model, decreased MFAP4 concentration was also an independent determinant of STEMI (OR = 0.395, 95% CI 0.174-0.895, p = 0.026).

Diagnostic and prognostic value of circulating MFAP4 levels
In order to differentiate AMI from UA, ROC was constructed and the area under the curve (AUC) of MFAP4 was 0.761 (p < 0.001, 95% CI 0.687-0.835). The optimal cut-off point of 0.117 ng/ml was selected with the higher sensitivity (96.7%) and specificity (57.8%) (Fig. 3A). Additionally, in differential diagnosis between STEMI and non-STEMI, ROC analysis showed that the AUC was 0.653 (p = 0.019, 95% CI 0.519-0.788) (Fig. 3B), indicating that MFAP4 may be a potential biomarker.
According to the cut-off value (0.117 ng/ml), ACS patients were divided into two groups: the low-MFAP4 group (MFAP4 < 0.117 ng/ml, n = 60) and the high-MFAP4 group (MFAP4 ≥ 0.117 ng/ml, n = 88). The median follow-up was 165 (147, 199) days, and in Kaplan-Meier survival analysis, patients of lower MFAP4 levels had an increased event-free survival rate (p = 0.009) (Fig. 4A), and when this analysis was performed in the STEMI group, the same significant outcome was obtained (p = 0.016) (Fig. 4B). ROC curve analysis identified that the AUC for the baseline MFAP4 for the prediction of MACE in ACS was 0.679 (95% CI 0.554-0.805, p = 0.015) (Fig. 5).

Discussion
This is the first study to date evaluating the association between circulating plasma MFAP4 levels and ACS and its clinical outcomes. In this study, we presented a significantly decreased MFAP4 concentration in STEMI, compared with non-STEMI and UA, which was correlated with coronary stenosis and myocardial injury markers, and to our surprise further demonstrated that higher MFAP4 levels prospectively predict the development of MACE in patients with ACS. Human MFAP4 was mapped to the 17p11.2 chromosome and is first reported in Smith-Magenis syndrome [10]. It belongs to a member of the microfibrillar-associated proteins (MFAPs) family, and the majority of MFAPs are traditionally involved in the organization of elastic fibers by specifically binding to tropoelastin [4]. Similarly, MFAP4, ubiquitously distributed in extracellular matrix, was found to promote tropoelastin self-assembly, involved in proper elastic organization, and specifically bind fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine [11]. MFAP4 mRNA relative transcript levels are highest in the heart, but the greatest abundance of MFAP4 is located in elastic fibers within blood vessels and surrounding connective tissue; cardiomyocytes do not contain MFAP4 [3]. Although its biological functions are not fully investigated, MFAP4 probably contributed to many disease conditions. As reported, in pulmonary physiology MFAP4 was involved via modulation of airway smooth muscle cells, participation in maintaining the integrity of the alveolar septa and interaction with lung surfactant protein D [12]. Since the presence of soluble desmosine is an indicator of elastic fiber degradation, plasma MFAP4 levels has been a potential indicator for chronic obstructive pulmonary disease (COPD) severity [13]. Also, MFAP4 has been proposed as markers of tumor progression and is linked to breast cancer [14], serious ovarian cancer [15] and neuroblastoma [16]. Additionally, initially identified as a biomarker of liver fibrosis, MFAP4 was subsequently confirmed its diagnostic capability in hepatitis C virus or alcohol-induced liver fibrosis [17]. Regarding artherosclerotic diseases, the expression of MFAP4 was reported to increase in vascular smooth muscle cells within large arteries, especially be activated and released following injury [2]. Previous basic studies on Mfap4-deficient mice have confirmed that MFAP4 participated in vascular smooth muscle hyperplasia and vascular remodeling in peripheral artery disease (PAD), and subsequent clinical research revealed that in symptomatic PAD patients upper tertile MFAP4 was significantly associated with cardiovascular death, but a surprisingly decreased risk of occlusion of reconstructed vessel [18]. Similarly, in our study, the baseline plasma MFAP4 level was negatively associated with coronary stenosis while increased MFAP4 levels prospectively predict the development of MACE in ACS (see Additional file 1).
A possible explanation of our present findings of negative relationship between MFAP4 levels and coronary stenosis in ACS may attribute extracellular matrix remodeling during plaque rupture. As one of the determinants of ACS severity [19], the integrity of the fibrous cap over plaque and its resistance to rupture depends critically on the content of extracellular matrix collagen and elastic fibers [20,21]. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine. Site-directed mutagenesis disclosed residues Phe 241 and Ser 203 in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Besides, the same study found that MFAP4  actively promotes tropoelastin self-assembly [11]. Thus, low MFAP4 levels are likely indicative of fibrous cap instability, which help explain why MFAP4 levels were relatively lower in STEMI compared with non-STEMI and UA. In agreement with our findings, Stakhneva E et al. found that MFAP4 levels increased only in stable atherosclerotic plaques in the fat deposition and fibrosis stages, but not in the fibrosis and calcification stages, nor in unstable atherosclerotic plaques of the necrotrophic-dystrophic type [22]. Contrary to the opposite trend between MFAP4 and coronary stenosis, our study found a significant decrease in asymptomatic survival in the high MFAP4 group, which may suggest that MFAP4 plays different roles in the disease process. As illustrated by Helle, MFAP4levels were particularly lower in patients with stable atherosclerotic disease compared with STEMI and non-STEMI [3]. With gradually aggravated myocardial ischemia, increased MFAP4 probably help alleviate vascular injury and subsequent atherosclerotic changes, and contribute to neovascularization of ischemic myocardium. Vascular smooth muscle cells (VSMCs) activation and phenotypic switching are critical for vascular remodeling wherein integrins are of particular importance [23]. MFAP4 serves as an integrin ligand localized to extracellular matrix fibers in the vascular wall, which has been found to regulate integrin αVβ3-induced VSMC proliferation and migration, and to accelerate neointimal hyperplasia after vascular injury [2]. Culture of isolated human saphenous veins for 14-21 days showed that MFAP4 was present in the vessel wall and that its deposition was induced in the area directly below the growing neointima [2], demonstrating that MFAP4 concentrations may parallel intimal neointima. We therefore suggest that MFAP4 may increase the risk of restenosis after coronary intervention, which explains why high levels of mfap4, a protective factor in ACS, was a predictor of MACE events in the present study. Further experiments are needed to monitor MFAP4 concentrations over longer cycles after the onset of ACS.

Limitations
Several limitations of present study need to be considered. First, relatively small cohort and limited followup in this study limited further valuable information on the role of MFAP4 in ACS. Second, healthy controls were not included and other biomarkers were not sampled in this study. Third, there was no follow-up data on